Novel Single-Enzyme Assisted Dual Recycle Amplification Strategy for Sensitive Photoelectrochemical MicroRNA Assay.

Herein, a novel single-enzyme assisted dual recycle amplification strategy based on T7 exonuclease (T7 Exo) and strand displacement reaction (SDR) was designed to fabricate photoelectrochemical (PEC) biosensor for sensitive microRNA-141 (miRNA-141) detection with use of laminar bismuth tungstate (Bi2WO6) as photoactive material. Compared with traditional enzyme assisted dual recycle amplification strategy, the presented method could effectively refrain enzyme interference reaction, reduce environmental sensitivity and save cost. Here, hairpin DNA1 (H1) decorated on magnetic beads (MB) hybridized with target miRNA-141 to form H1/miRNA-141 heteroduplex. With the introduction of hairpin DNA2 (H2) labeled SiO2 (H2-SiO2), SDR was triggered between H2-SiO2 and H1, thus miRNA-141 was displaced from H1/miRNA-141 heteroduplex and H1/H2-SiO2 duplex was formed, realizing the reuse of target. In the existence of T7 Exo, the H1/H2-SiO2 duplex was digested with the release ofoutput DNA-SiO2. To enhance the target conversion rate, H1-MB was intactly released and cycled, which could initiate more T7 Exo digestion and freed abundant output DNA-SiO2. Through such a process, a tiny miRNA-141 could induced substantial output DNA-SiO2, effectively improving the target amplification efficiency and detection sensitivity of PEC biosensor. Furthermore, Bi2WO6 was modified on electrode to provide a superior initial PEC signal due to its excellent electronic transformation capacity. With the introduction of output DNA-SiO2, the hairpin structure of H3 on electrode was opened, making SiO2 close to the electrode surface, which significantly decrease the PEC signal. This work firstly established a PEC biosensor featured a single-enzyme assisted dual recycle amplification process for sensitive detection of biomarkers.